Cholersterol esters are the storage form of cholesterol. The distribution between free and esterified cholesterol may be an important determinant of cholesterol homeostasis. The rate of cholesterol esterification is determined by the activity of the enzyme acyl CoA: cholesterol acyltransferase. We propose to study the mechanisms of regulation of this enzyme in detail. Initially in vitro studies will be utilized to elucidate the mechanism of the rapid response of this enzyme to steroids. To do this, techniques for addition to steroids to microsomes have been devised as has a method of kinetic analysis. The importance of membrane bulk phase lipid fluidity as well as sterol domains in the process will be assessed by electron spin resonance and possibly fluorescence polarization. These techniques will be extended to livers whose sterol content has been affected by physiologic stimuli. The importance of other reactants (acyl CoA and cholerterol esters) will also be studied as will the possibilities that the enzyme is partially latent, can be activated by covalent modification and can be increased or decreased in quantity. Purification of the enzyme will be done to further study its regulation. The physiologic implications of alteration of this enzyme's activity are being studied in isolated liver and in intestine. Finally we propsed to conclude our studies on the characterization and regulation of intestinal HMG-CoA reductase.